Molecular diagnosis generally refers to nucleic acid analysis for detecting infection sources, genetic diseases, cancer and genetic variations of patients. It identifies presence of diseases or pathogens and likelihood of genetic disorders by detection or quantification of genetic materials from samples derived from human body such as blood, urine and saliva. The molecular diagnosis process generally includes in vitro amplification of nucleic acid molecules by amplification reactions such as polymerase chain reaction (hereinafter referred to as “PCR”) and real-time PCR.
The most predominant process for nucleic acid amplification known as PCR is based on repeated cycles of denaturation of double-stranded DNA, followed by oligonucleotide primer annealing to the DNA template, and primer extension by a DNA polymerase (Mullis et al. U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al, (1985) Science 230, 1350-1354). Oligonucleotide primers used in PCR are designed to anneal to the opposite strand of DNA templates. The primers are extended by nucleic acid polymerase and the resulting extension product is served as a template strand for another primer in following reaction rounds. PCR amplification results in exponential increase of DNA fragments. For PCR, two primers, polymerase and nucleic acid template are typically utilized.
Nucleic acid amplification is a pivotal process for a wide variety of methods in molecular biology, such that various amplification methods have been proposed: LCR (Ligase Chain Reaction), GLCR (gap filling LCR), Q-beta (Q-beta replicase amplification), SDA (Strand Displacement Amplification), 3SR (self-sustained sequence replication), NASBA (Nucleic Acid Sequence-Based Amplification), TMA (Transcription Mediated Amplification) and RCA (Rolling-Circle Amplification). Novel and improved processes to a typical PCR protocol have been proposed.
Multiplex PCR was developed to simultaneously amplify multiple targets in a reaction and real-time PCR to qualitatively and quantitatively analyze amplification reactions of a target nucleic acid in a real-time manner.
Real-time PCR is one of PCR-based technologies in which a target nucleic acid is amplified together with measurement of the amplification reaction in a real-time manner, thereby detecting the target nucleic acid.
According to the typical PCR technologies, the reaction resultant is taken out after completion of amplification and then the presence or concentration of a target nucleic acid is measured. Unlikely, real-time PCR technologies are capable of is determining the presence and concentration of a target nucleic acid in a real-time manner. Real-time PCR technologies generally use labeled probes to be hybridized with a target nucleic acid. Methods involving hybridization between a labeled probe and a target nucleic acid include Molecular beacon method (Tyagi et al, Nature Biotechnology 14:303 (1996)), Hybridization probe method (Bernad et al, Clin Chem 46:147 (2000)) and Lux method (U.S. Pat. No. 7,537,886). TaqMan method widely used in the art utilizes hybridization of a dual-labeled probe and its cleavage reaction by 5′ nuclease activity of DNA polymerase (U.S. Pat. Nos. 5,210,015 and 5,538,848).
The real-time detection methods are a homogeneous assay to perform amplification reaction and detection in a single tube, such that they require no additional operation and are free from carry-over contamination.
Enzymes are generally unstable in a liquid form at room temperature. Therefore, enzymes are usually stored in a lyophilized form or in a liquid form using stabilizer at −20° C. Although various storage strategies were proposed, activities of enzymes are very likely to be deceased due to frequent thawing and handling at room temperature.
Primers may form primer dimers by intra- or inter-strand primer annealing when stored in an aqueous environment, particularly at room temperature. In general, where a PCR reaction mixture is kept to stand for about 30 min at room temperature and then used for PCR amplification reaction, the production of a typical PCR product may be inhibited and sometimes may not be made. Furthermore, where a PCR reaction mixture is kept to stand for several hours to days at room temperature and then used for PCR amplification reaction, the production of a typical PCR product is very unlikely to occur.
Hitherto, most of PCR-based diagnosis products are supplied in the liquid form and have to be stored at −20° C., which are responsible for higher costs for transportation and storage.
A PCR process applied to various diagnosis technologies requires very elaborative techniques. The conventional PCR process is performed in such a manner that primers, polymerase, dNTPs, buffer and magnesium chloride are successively introduced into microtubes or multi-wall plate to prepare a reaction mixture and then nucleic acid template is introduced for amplification reaction. Some reagents such as polymerase and dNTPs have to be stored at −20° C. and other reagent such as primers and probes have to be stored at low temperature. As such, PCR-based diagnosis experimentations require dispensing small quantity of various materials which should be stored and handled under stringent conditions. Since the PCR-based diagnosis methods involve complicated and elaborative steps, very skillful technicians are demanded. It has been well known to one of skill in the art that PCR process is likely to produce false negative results due to experimentation errors and false positive results due to carry-over contamination.
To overcome such shortcomings, a PCR master mix in the dried form was suggested. The drying technologies are classified to two approaches. In the first approach, air drying at room temperature or high temperature under atmospheric pressure is performed. The second approach is lyophilization method in which samples are frozen and solvent molecules in the frozen samples are removed by sublimation. The lyophilization method comprises a freezing step and a drying step, which can remove solvent in a solid state to minimize structural changes during drying
Lyophilization involves freezing a formulation, preferably by quick-frozen process. The frozen sample is then subject to sublimation of solvent in the frozen state at below freezing-point under high vacuum (a primary drying). Afterwards, a residual solvent is additionally removed with successively elevating temperatures (a secondary drying), providing a product in the form of crystal or powder. The final lyophilized product (lyophilizate) is in the form of porous cakes having the same shape and size as generally frozen materials.
Since the properties of lyophilizates are greatly affected by the shape and structure of cakes, high-quality lyophilizates require cakes with favorable shapes and structures. The lyophilizate cakes are required not to be disrupted because disrupted cakes are scarcely restored to possess their initial activities by reconstitution (rehydration). The physical structure of lyophilizate cakes should not be loose or soft.
The stability and post-reconstitution activity of lyophilizates are dependent greatly on properties of materials to be lyophilized and process of lyophilization. For successful lyophilization, various stabilizers and stabilization methods were proposed. Frank et al. suggested that carbohydrates as a cryoprotectant would improve stability and storage quality of lyophilizates (U.S. Pat. No. 5,098,893). However, the method has shortcomings in the senses that a drying step is performed at room temperature or elevated temperature around atmospheric pressure. American Type Culture Collection, Inc. commercialized lyophilized DNAs containing lactose as a cryoprotectant in the early 1980s. U.S. Pat. No. 5,955,448 discloses a stabilizing method in which biological samples having free amino, imino or guanidino side chains are incubated with non-reducing carbohydrate additive and inhibitor to Maillard reaction to prevent aggregation between free amino groups and reactive carbonyl groups. De Luca et al. proposed PCR lyophilized composition containing cellobiose as stabilizers (U.S. Pat. Appln. Pub. No. 2012/0064536). Perry et al. reported dried compositions containing fluorescent dye-linked nucleotides and Taq DNA polymerase substantially without glycerol (U.S. Pat. No. 7,407,747). Rajeev et al. suggested rotavirus vaccine compositions containing sucrose and glycine as additives (U.S. Pat. No. 8,795,686) and Fumitomo et al. suggested a lyophilization composition for reverse transcriptase containing trehalose, nucleic acid and metal salt (U.S. Pat. No. 5,935,834).
The real-time detection methods demand not only primers for amplification but also labeled primers or probes for detection. A PCR reagent mixture of multiplex PCR comprises at least two primer pairs for simultaneously amplifying multiple targets. In a composition for multiple target nucleic acid amplification such as multiplex PCR, oligonucleotides are contained at higher proportion or concentration in proportion to the number of the target. For simultaneously detecting multiple targets, the combination of real-time PCR and multiplex PCR (real-time multiplex PCR) has been suggested.
The compositions containing high-concentrated oligonucleotides are likely not to form lyophilizate cakes. Even though lyophilizate cakes are formed, the compositions containing high-concentrated oligonucleotides suffer from lyophilizate cake with unfavorable shapes and structures.
Accordingly, there is an urgent need to develop a novel method for lyophilizing compositions containing high-concentrated oligonucleotides for multiple target nucleic acid sequence amplification reaction.
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entirety are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.